Chemical reactivity and biological activity of diketene

The alkylating potential of diketene (4-methylene-2-oxetanone), the basic unit of many derivatives of pesticides, chemicals, pharmaceuticals, and dyestuffs, was investigated kinetically. The nucleophile 4-( p-nitrobenzyl)pyridine (NBP), a trap for alkylating agents with nucleophilic characteristics similar to DNA bases, was used as an alkylation substrate. The alkylation reactions were performed in water/dioxane solvent mixtures. To gain insight into the effect of the hydrolysis of diketene on its alkylating efficiency, alkylation and competing hydrolysis were studied in parallel. Conclusions were drawn as follows: (i) Although diketene, unlike other four-membered ring lactones, is inactive as a carcinogen in experimental animals, it shows an alkylating potential of about 2 orders of magnitude higher than beta-propiolactone or beta-butyrolactone, which are classified as possibly carcinogenic to humans by the IARC. (ii) The reactivity of diketene as an alkylating agent is enthalpy-controlled. (iii) The fact that the hydrolysis reaction of diketene is slightly faster than those of other four-membered ring lactones shows that diketene is more efficient than beta-propiolactone or beta-butyrolactone as an alkylating agent, since the hydrolysis of this species poses less competition to the alkylation reaction. (iv) Diketene undergoes acyl fission in the alkylation reaction, which results in an amide bond in the NBP-diketene adduct. The lability of the amide bond as opposed to the amine bonds formed by beta-propiolactone and beta-butyrolactone could be one of the differential factors responsible for the lack of carcinogenicity of diketene. (v) Ab initio calculations of the energy barriers help to understand the unusual reactivity of diketene.     

Hemorrhagic activity of Bothrops venoms determined by two different methods and relationship with proteolytic activity on gelatin and lethality

The changes in hemorrhagic activity, proteolytic activity on gelatin and the lethal potency of four Bothrops venoms treated at different pH values or with EDTA were studied. Venoms from B. alternatus, B. jararaca, B. moojeni and B. neuwiedii of Argentina were preincubated at pH 5.8, 5.1 or 3.8 or with EDTA and the hemorrhagic activity expressed as size of the hemorrhagic lesion or as the amount of hemoglobin extracted, the proteolytic activity on gelatin and the lethal potency were determined. Although the MHDs recorded in rats were 19–56 fold higher than those recorded in mice, the A₅₅₀ extracted per gram of hemorrhagic haloes was very similar in rats or mice independent of the venom dose. Inhibition of proteolytic activity after preincubation at pH 5.1 or 3.8, agrees with the decreased amount of hemoglobin extracted from the hemorrhagic haloes, and with the increase in mean survival time after the i.p. injection to mice. Preincubation with EDTA resulted in 80% inhibition of hemorrhagic activity of B. jararaca venom and complete inhibition with the other Bothrops venoms tested. Measurement of the amount of hemoglobin extracted gives significant information in comparative studies, not available by measurement of the size of hemorrhagic haloes.     

Genetic Toxicology of Dental Composite Resin Extracts in Somatic Cells In Vivo

Abstract: The aim of this study was to assess the potential genetic toxicity associated to nine aqueous extracts from dental composite resins (Charisma, Fill Magic, Fill Magic Flow, Durafill, TPH Spectrum, Concept, Natural Look, Filtek Z250 and Filtek P60) and one random extract. Homologous mitotic recombination, point and chromosomal mutation effects were determined in somatic proliferative cells of Drosophila melanogaster exposed to aqueous extracts of the clinically used composites. Reproducible increases in clone mutant spot frequencies induced by diluted extract of Fill Magic Flow were observed. These increments were exclusively associated to the induction of homologous recombination – a genetic phenomenon involved in the loss of heterozygosis. The other eight composite resins and the random extract had no statistically significant effect on total spot frequencies – suggesting that they are non‐genotoxic in the somatic mutation and recombination test assay, which agrees with the applications they have in dentistry. These findings – supported by numerous studies showing a positive correlation between carcinogenicity in man and genotoxicity in the Drosophila wing spot test – point to the potential risks some composite resins pose to the health of patients and dentistry personnel.     

<Emphasis Type="Italic">EGFR</Emphasis> Amplification and Sensitizing Mutations Correlate with Survival in Lung Adenocarcinoma Patients Treated with Erlotinib (MutP-CLICaP)

Background

Non-small cell lung cancer (NSCLC) has a 5-year survival of 5–16%. Epidermal growth factor receptor (EGFR) mutations, in most cases, confer sensitivity to EGFR tyrosine kinase inhibitor (TKI) therapy. Nonetheless, it is still unclear why clinical outcomes vary among patients with identical EGFR mutations. The amplification of the EGFR gene (EGFRamp) may play a significant role.

Objective

Compare the complete (CR) and partial response (PR) rates, overall survival (OS), and progression-free survival (PFS) in Hispanic patients with lung adenocarcinoma treated with erlotinib with EGFR mutations (L858R or exon 19 deletion [Del19]) with and without concomitant EGFRamp.

Patients and Methods

Seventy-two EGFR-positive lung adenocarcinoma patients of Hispanic origin, who underwent first-line treatment with erlotinib, were evaluated for EGFRamp by fluorescence in situ hybridization (FISH). The clinical outcomes were analyzed according to EGFR mutations and EGFRamp status.

Results

30.6% of samples showed EGFRamp, more frequently present in patients with Del19 (p?=?0.05). Patients with EGFRamp had a longer PFS (in months) [(28.5, 95% CI 22.3–34.6) vs. (11.0, 95% CI 8.2–16.7); p?=?0.002] and OS [(37.8, 95% CI 30.9–44.7) vs. (27.1, 95% CI 12.8–41.3); p?=?0.009] than those without. EGFRamp significantly influenced the response to erlotinib (p?=?0.0001). EGFRamp+/Del19 had a longer OS, 37.8 (95% CI 31.0–44.6), compared to EGFRamp+/L8585R, 27.5 (95% CI 12.4–42.5) (p?<?0.001) and longer PFS (p?=?0.043).

Conclusion

Among Hispanic patients, EGFRamp was present in 30% of patients with EGFR mutations. EGFR mutations and EGFRamp are associated with better OS, PFS, CR, and PR to erlotinib and, hence, could aid in the correct selection of patients that benefit from EGFR TKI treatment.

     

Complex regulation of the fibroblast growth factor-binding protein in MDA- MB-468 breast cancer cells by CCAAT/enhancer-binding protein beta

The fibroblast growth factor-binding protein (FGF-BP) binds and activates fibroblast growth factors in the extracellular matrix, and can have a rate-limiting role in tumor angiogenesis. Here we demonstrate high levels of FGF-BP expression in invasive human breast cancer, relative to normal breast and in situ carcinoma, and in MDA-MB-468 human breast cancer cells. In these cells, FGF-BP was up-regulated by treatment with epidermal growth factor (EGF), dependent on protein kinase C and p38 mitogen-activated protein kinase signaling. Mutational analysis revealed that the activator protein 1 and CCAAT/enhancer binding protein (C/EBP) sites on the FGF-BP gene promoter were required for the EGF effect, whereas deletion of the C/EBP site resulted in a significant increase in promoter basal activity indicating a basal repressive control mechanism. These data suggest that the C/EBP site is a central regulatory element for the regulation of FGF-BP promoter activity in MDA-MB-468 cells. We found that MDA-MB-468 cells express high endogenous levels of both the activating (LAP) and repressive (LIP) isoforms of C/EBPbeta. Overexpression of C/EBPbeta-LAP in MDA-MB-468 cells resulted in a large 80-fold increase in FGF-BP promoter basal activity, which was reversed by coexpression of LIP. Gel-shift analysis revealed that four LIP- and LAP-containing complexes (a-d) bind to the C/EBP site. DNA binding of the LIP and LAP-containing c complex and the b complex in the presence of EGF was modulated by inhibition of p38 mitogen-activated protein kinase, suggesting a role for these complexes in the EGF induction of the FGF-BP promoter. This study suggests that along with its well-defined role in mammary gland development, C/EBPbeta may well play a role in the pathology of breast cancer, in particular in the control of angiogenesis in the invasive phenotype.     

Effect of an antioxidant functional food beverage on exercise-induced oxidative stress: A long-term and large-scale clinical intervention study

The efficacy of long-term intake of a novel functional food supplement Funciona™ containing vitamins and juiced fruits was evaluated in order to assess the net effect of physical activity and antioxidant potentials in healthy older adult population. The long-term (2 years) and large-scale (400 older adult subjects) interventional study was based on both moderate-intensity exercise practice and concurrent supplementation. Sustained exercise-induced oxidative stress as reflected in significantly increased blood thiobarbituric acid-reactive substances (TBARS) (+15%), protein carbonyl groups (PC) (+18%) and oxidized glutathione (GSSG) (+112%) concentrations, and leukocyte 8-OHdG contents (23%). Exercise decreased the reduced/oxidized glutathione (GSH/GSSG) molar ratio (−43%) and plasma vitamin C levels (−22%). Supplementation with Funciona™ was significant in preventing oxidative damage to lipid, protein and DNA, and normalizing blood GSSG, GSH/GSSG and vitamin C levels. Thus daily intake of the antioxidant functional beverage counteracts the exercise-induced oxidative stress in free-living older subjects, and might be necessary to restore impaired antioxidant balance due long-term regular exercise.     

Prevalence of extended-spectrum beta-lactamase and carbapenemase-producing bloodstream isolates of Klebsiella pneumoniae in a tertiary care hospital

To improve prescribing of empiric therapy, the local molecular epidemiology of extended-spectrum beta-lactamases (ESBLs) and Klebsiella pneumoniae carbapenemases (KPCs) in bloodstream isolates of K. pneumoniae were evaluated. Isolates resistant to third generation cephalosporins were screened phenotypically for ESBLs and carbapenemases, and subsequently confirmed by PCR for the presence of ESBL (bla_TEM, bla_SHV and bla_CTX-M) and carbapenemase (bla_KPC, bla_VIM, bla_NDM and bla_OXA-48) genes. Hydrolytic activity (functional gene expression) was quantified using a nitrocefin degradation assay and correlated to ceftazidime or meropenem MIC. Clonality was assessed by repetitive element-based PCR. Beta-lactamases were functionally expressed in 13 isolates (15.5%); 7 (53.8%) harboured bla_CTX-M-15 and 6 (46.2%) carried the bla_KPC-2 gene. Correlation of hydrolytic activity to MIC yielded a coefficient of 98% for isolates expressing ESBLs alone and 56% for carbapenemase producers. Four unique ESBL-expressing clones and five carbapenem-resistant clones were identified. All 13 resistant isolates were susceptible to ceftazidime/avibactam (MIC ≤ 8/4 mg/L).     

<Emphasis Type="Italic">BRCA1</Emphasis> deficiency is a recurrent event in early-onset triple-negative breast cancer: a comprehensive analysis of germline mutations and somatic promoter methylation

Purpose

BRCA1 germline mutation is closely associated with triple-negative breast cancer. BRCA deficiency leads to impaired DNA repair and tumor development, and understanding this deficiency, in both hereditary and sporadic scenarios, is of great clinical and biological interest. Here, we investigated germline or somatic events that might lead to BRCA1 impairment in triple-negative breast cancer. We also analyzed the clinical implications associated with BRCA deficiency.

Methods

Next-generation sequencing for the BRCA1/2 genes and multiplex ligation-dependent probe amplification (MLPA) for the BRCA1 gene were performed for mutation screening. A customized bisulfite next-generation sequencing approach was used for assessing BRCA1 promoter methylation status in tumor tissue.

Results

A total of 131 triple-negative cases were assessed, and germline pathogenic variants were detected in 13.0% of all cases and in 26% of cases diagnosed in young women. Most germline pathogenic variants (88.2%) occurred in the BRCA1 gene. BRCA1 promoter hypermethylation was detected in 20.6% of tumors; none of these tumors were in BRCA1/2 pathogenic variant carriers. BRCA1 impairment by either germline or somatic events was significantly more frequent in young women (55% in those ≤ 40 years; 33% in those 41–50 years; 22% in those > 50 years of age) and associated with better overall and disease-free survival rates in this group of patients.

Conclusions

BRCA1 deficiency was recurrent in early-onset triple-negative breast cancer in Brazilian patients and associated with improved survival. With the new treatment modalities being investigated, including poly (ADP-ribose)-polymerase (PARP) inhibitor therapy, our results suggest that a significant proportion of young women with this subtype of tumor might benefit from PARP inhibitor treatment, which warrants further investigation.